Abstract 1477: Identification and Functional Analysis of Protein Interaction Partners of the Estrogen Receptor Alpha in the Human Heart
Background and aims: Estrogens (E2) are important regulators of physiological and pathological processes in various target tissues, including the human heart. Most of the biological actions of E2 are mediated by nuclear steroid receptors, the estrogen receptor alpha (ERα) and beta (ERα), which function as hormone-inducible transcription factors. ERs act in concert with other regulatory elements to mediate estrogenic effects. So far, only few cofactors of ERs have been described in the human heart. To gain a better understanding of E2-mediated ERα action in the human heart, we identified and functionally analyzed the interaction partners of ERα.
Methods: The yeast two hybrid screen (YTH) was performed in the presence of E2 with ERα as bait in a human heart cDNA library. Co-immunoprecipitation analysis (Co-IP) with human atrial proteins was carried out to confirm the interaction in a mammalian system. Immunofluorescence (IF) methods were employed on human atrial tissue samples as well as in cotransfected AC16 cells to analyze the co-localisation of ERα and its binding partners. Promoter reporter assays were performed to show the regulation of the identified interaction partner by ERα.
Results: Yeast two hybrid screening of a human heart library revealed that ERα interacts with the atrial natriuretic peptide precursor A (NPPA). Retransformation experiments showed that NPPA can interact with ERα full-length and the ERα-EF domain in an E2-dependent manner. The LxxLL-motif of NPPA was found to interact with the ERα-EF domain in yeast. The interaction of NPPA and ERα was also confirmed using proteins derived from human atrium by Co-IP. IF analysis showed that E2 treatment of AC16 cells led to an increased translocation of NPPA and ERα in the nucleus of AC16 cells and also their co-localization there. Furthermore, expression analysis with NPPA luciferase promoter constructs showed a significant increase in the promoter activity in the presence of E2/ERα.
Conclusion: We showed the interaction of ERα and NPPA in an E2-dependent manner. Our findings suggest that the E2-activated ERα and NPPA complex binds to specific regions on the NPPA promoter leading to transcriptional regulation of the NPPA gene.